The Ets family member ERG has been shown to be frequently over-expressed in prostate cancer. Perhaps more strikingly, ERG and the Ets protein ETV1 have recently been shown to be the targets of chromosomal translocations with TMPRSS2 which are observed in 80% of prostate cancer patient samples. Indeed, the expression of TMPRSS2 is androgen regulated, resulting in over-expression of ERG or ETV1 in these prostate cancers. More extensive studies of the effects of over-expression of ERG in prostate cells have recently been carried out. Two studies have shown that over-expression of ERG results in increased invasion. Knockdown of ERG in the prostate cancer cell line VCaP has been shown to inhibit invasion. Microarray analysis shows that ERG knockdown in VCaP cells results in increased expression of genes associated with differentiated luminal prostate epithelial cells, suggesting that one of the roles of ERG overexpression in prostate cancer may be to block differentiation. DNA binding mediated by the Ets domain is essential for Ets protein gene regulation and certainly essential for the dysregulation mediated by over-expressed ERG. Indeed, DNA-binding has been shown to be essential in the context of the fusion between EWS and the Ets protein FLI-1, a close relative of ERG. The EWS-FLI1 fusion is found in a high percentage of Ewing's sarcoma patients. Point mutations in the Ets domain of the EWS-FLI1 fusion protein which impair DNA binding disrupt the transforming ability of EWS-FLI1. The importance of DNA-binding to the function of these proteins strongly suggests that inhibiting the DNA binding activity of ERG may be a mechanism to treat prostate cancer as well as other cancers where the activity of ERG plays a key role. Our goal is to develop small molecule inhibitors of this interaction as probes to test this hypothesis and lay the groundwork for the development of targeted therapies against ERG. To that end, we are proposing to use high throughput screening (HTS) to identify initial lead compounds which inhibit this protein-DNA interaction. We will use a well-validated fluorescence polarization assay to screen the MLPCN library of compounds. The most active compounds will be verified using an HTRF assay and NMR confirmation of binding. Specificity will be assessed by screening against an unrelated protein-DNA interaction as well as screening against other Ets family members. The compounds will subsequently be optimized to increase binding affinity for the Ets domain by structure-aided drug design approaches combined with standard medicinal chemistry. The most potent compounds will be tested in appropriate prostate cancer cell lines harboring ERG translocations and, if feasible, in appropriate mouse models of prostate cancer.